We propose to develop advanced fluorogenic probes that use the principle of RNA- templated chemistry to report on the presence of specific disease-relevant RNAs in solution and in cells. The analysis of cellular RNAs is increasingly important to diagnosis, treatment and prognosis of disease; however, current clinical methods for detection and identification of native RNAs are laborious and expensive, and cannot be applied in intact cells or tissues. Our recent work has developed designs for fluorogenic probes that offer the highest sequence specificity and lowest background of any probes yet reported, and thus offers much promise for solving these problems. However, further development is required in lowering background signal for the rarest targets, and for addressing folded target structure. Moreover, as with most DNA probes, the issue of probe delivery needs to be addressed (for whole-cell applications) by taking advantage of recently developed next-generation delivery agents. Our proposed work will address these issues by (a) developing novel chemistries and strategies that focus and intensify signal and lower background markedly; (b) exploring new molecular transporters, based on oligoguanidine-lipid conjugates, for enhancing cellular uptake, and by (c) collaboratively studying the detection and identification of type A influenza RNAs in vitro and directly in cells. In the near term covered by this proposal, this work is significant for its development of new chemistries that could lower background and enhance signal for RNA detection markedly. Moreover, it will address some of the most important and widely recognized problems that are faced in the nucleic acids field, including the problems of target accessibility and cell permeability. The work is novel as well: we will develop previously unreported strategies for signaling including template-triggered fluorophore release and capture, and templated chemiluminescence, as well as previously untested molecular strategies for cell uptake. In the longer term, the development of low-background fluorogenic templated probes could allow for unprecedented and widespread applications in medicine.